Epigenetic marks such as DNA methylation play important roles in mammalian development and the onset of disease. Our ability to study methylation in complex tissues is limited because existing tools measure the average methylation of a large number of cells. Since most tissues contain many different cell types, these bulk measurements are inaccurate. We hope to create an entirely new way to analyze methylation in complex tissues. We propose MethylMap, a high-throughput technology that combines multiplex amplification, sample-specific DNA barcodes, and next- generation sequencing to analyze methylation in laser capture micro-dissected cells. Our research plan consists of two specific aims. First, we will demonstrate the multiplexed amplification and single-molecule sequencing of 5000 promoters from a single sample of bisulfite treated DNA. We will use this technology to catalogue epigenetic mutations that arise during tumorigenesis. Next, we will use the 454 FLX sequencing platform to bisulfite sequence 200 promoters from laser capture microdissected cells arrayed in 96 well plates (or 19,200 promoters per plate). Each sample will be barcoded with a DNA tag to connect the measured methylation pattern with the location of the cells. We will build on preliminary results that demonstrate 1) multiplexed amplification of 90 loci from a single sample, 2) deep single molecule bisulfite sequencing of the MLH1 promoter in tumors using the 454 Life Sciences FLX sequencer, and 3) the use of sample-specific DNA barcodes with next-generation sequencing technology. MethylMap will have many applications: it will be used to molecularly classify cells in complex tissues, to determine the lineages of somatic cells, and to probe the role of aberrant methylation in tumorigenesis. We believe MethylMap will become an important tool for scientists interested in development, tissue homeostasis, and tumor biology. PUBLIC HEALTH RELEVANCE: DNA methylation is an important mechanism used by the cell to control gene regulation. Recent studies have shown that DNA methylation is critical for mammalian development, and if DNA methylation is misregulated, it can lead to cancer. Current tools to study DNA methylation are inadequate because they cannot effectively analyze complex tissues that contain many different types of cells. We propose to develop MethylMap, a technology that will make it possible to analyze the methylation patterns of cells in complex tissues for the first time. This tool will provide new insights into how tissues form, and how cancers arise.